The induction of tumor vasculature, known as the 'angiogenic switch', is a rate-limiting step in tumor progression. Most functional studies have focused on the responses of endothelial cells to pro-angiogenic stimuli; however, there is mounting evidence that the supporting mural cells (smooth muscle cells and pericytes) play a key regulatory role in maintaining a mature, quiescent vasculature. In tumors, mural cell association with the endothelium is decreased and abnormal. Previous work has shown that restoration of functional inhibitory maturation to vasculature by Angiopoietin-1 inhibits tumor growth, suggesting that stabilization of tumor vessels may be a desirable therapeutic goal in the treatment of cancer. The hypothesis underly this work is that breast cancer cells functionally alter mural cell and endothelial cell contacts and subvert the mural cell from its normal anti-angiogenic role to a vessel-promoting role as part of the angiogenic switch. Paracrine interactions between endothelial cells, mural cells, and breast cancer cells will be studied using in vitro membrane and spheriod models that mimic the organization of the blood vessel wall, as well xenograft models with modified mural cells, in order to address three specific aims. Aim 1 will identify critical alterations in mural cell function in response to breast cancer cells that may contribute to the maturation defect exhibited by the tumor vasculature. Aim 2 will investigate the ability of tumor cells to activate matrix metalloproteases specifically in mural cells as part of the acquisition of a pro-angiogenic functional state. Aim 3 will address whether the differentiation utilization of specific sphingosine-1-phosphate receptors plays a role in the tumor-induced maturation defect and activation of mural cells. Together, these studies will i) provide proof-of-principle that tumors can subvert the function of normally inhibitory mural cells to a tumor-promoting state, and ii) identify pivotal molecular players involved in these activities to serve as targets for future mural cell-directed therapies to restore quiescence to the vasculature.